Abstract
The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.
Highlights
Despite very significant efforts from many laboratories, the mechanism regulating the ATP synthesis resulting from oxidative phosphorylation in cells is not yet understood in vivo
Three different pathways led to ADP generation: 1) high myofibrillar ATPase activity occurred under saturating [ATP] of 1 mM; 2) compartmentalized adenylate kinase (AK) activity participated to ADP production from ATP and AMP; 3) compartmentalized creatine kinase (CK), ScCKmit in the mitochondrial intermembrane space and bound MM-CK to myofibrils and sarcoplasmic reticulum
The results of this study confirm that the regulation of mitochondrial respiration in oxidative muscle differs from that in glycolytic muscle [24, 26]
Summary
Despite very significant efforts from many laboratories, the mechanism regulating the ATP synthesis resulting from oxidative phosphorylation in cells is not yet understood in vivo. It has been proposed that in muscle cells, the different isoenzymes are functionally and/or structurally coupled either to sites of energy consumption (cellular ATPases, such as actomyosin ATPase and sarcoplasmic reticulum Ca2ϩ-ATPase) or sites of energy production (glycolysis and mitochondria) to facilitate energy transport in the cytoplasmic compartment with creatine (Cr) and PCr as diffusible intermediates According to this model, ADP produced by high free-energy demand in myofibrils is immediately rephosphorylated into ATP through the action of cytosolic CK, using the phosphoryl group from PCr, while Cr diffuses back to mitochondria to be rephosphorylated into PCr through the action of ScCKmit bound to the outer surface of the inner mitochondrial membrane. In the current study, we investigated the characteristics of the regulation of mitochondrial respiration and its stimulation by Cr in heart, soleus, and white gastrocnemius taken from wild-type and transgenic mice, by using the permeabilized fiber technique [7]
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